This project is designed to isolate human 20 alpha-hydroxysteroid dehydrogenase and purify the enzyme to homogeneity using affinity chromatography. The physico-chemical characteristics of the purified enzyme will be studied and crystallization will be attempted. The aim of the project is to extensively study the topography of the enzyme active site using affinity labeling analogues of progesterone. The methodology from this study, which used human term placenta as a readily available source of human enzyme, will be a foundation for isolation of 20 alpha-hydroxysteroid dehydrogenase from tissues whose enzyme content is less rich or whose availability is more limited. Comparison of structural features which are common (and different) to enzyme from other sources (reported or being studied in this laboratory) will be made. Detailed biochemical knowledge of enzymes regulating steroid hormone metabolism and elucidation of the topographical requirements for interaction of a steroid hormone with a macromolecular high affinity binding site should enhance our understanding of the mechanisms of steroid hormone action.